The present invention relates generally to methods of treating solid tumors. More particularly, the invention relates to the use of recombinant adeno-associated virus (rAAV) virions to deliver a plurality of selected genes to cancerous cells and tissue. The method provides for the introduction of a drug-susceptibility gene and a second gene capable of providing an ancillary therapeutic effect into solid tumor cells. The invention also relates to rAAV virions that contain DNA useful in the treatment of neoplastic disease.
Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of viral based systems for gene transfer purposes have been described, such as retroviral systems which are currently the most widely used viral vector systems for gene transfer. For descriptions of various retroviral systems, see, e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
A number of adenovirus based gene delivery systems have also been developed. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro. Adenovirus is easily produced at high titers and is stable so that it can be purified and stored. Even in the replication-competent form, adenoviruses generally cause only low level morbidity and are not associated with human malignancies. For descriptions of various adenovirus-based gene delivery systems, see, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; Rich et al. (1993) Human Gene Therapy 4:461-476.
The in vivo transfer of specific tumor suppressor genes, apoptotic genes, or genes that encode a particular toxic product to cancer cells, using such known gene delivery systems, will provide an attractive alternative to conventional avenues in the treatment of neoplastic disease. Such approaches are particularly indicated in the treatment of cancers that are refractive to conventional procedures such as surgery, radiotherapy and chemotherapy. In this regard, advances in molecular biology have identified a number of mechanisms that control cell growth and differentiation. Experimental treatments which specifically target these pathways using gene therapy are currently underway. Particularly, a number of approaches involving somatic gene therapy in cancer treatment have been investigated, including drug sensitization, genetic immunomodulation, normal tissue protection, gene replacement and antisense strategies. Gutierrez et al. (1992) Lancet 339:715-721, Anderson, W. F. (1994) Hum. Gene Ther. 5:1-2.
Of these approaches, drug sensitization has provided the most promising results to date. Drug sensitization involves the transfer of suicide genes (e.g., drug-susceptibility genes) to tumor cells to render those cells sensitive to compounds or compositions that are relatively nontoxic to normal cells. Moolten, F. L. (1994) Cancer Gene Ther. 1:279-287. Examples of suicide genes are thymidine kinase of herpes simplex virus (HSV-tk), cytochrome P450 (Manome et al. (1996) Gene Therapy 3:513-520), human deoxycytidine kinase (Manome et al. (1996) Nature Medicine 2(5):567-573) and the bacterial enzyme cytosine deaminase (Dong et al. (1996) Human Gene Therapy 7:713-720). Cells which express these genes are rendered sensitive to the effects of the relatively nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide (cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine kinase) or 5-fluorocytosine (bacterial cytosine deaminase). Culver et al. (1992) Science 256:1550-1552, Huber et al. (1994) Proc. Natl. Acad. Sci. USA 91:8302-8306.
The HSV-tk gene is the most widely studied drug-susceptibility gene. HSV-tk converts specific protoxic nucleoside analogues, such as acyclovir and ganciclovir, to monophosphate intermediates that are then phosphorylated by cellular kinases to provide potent DNA synthesis inhibitors. Cells capable of expressing HSV-tk are rendered extremely sensitive to the toxic effect of ganciclovir, whereas non-HSV-tk expressing cells are much less sensitive, resulting in a large therapeutic index. Tumor modeling experiments using gene delivery of HSV-tk have demonstrated complete regression of established tumors and long-term animal survival, even though only a portion of the tumor cells were actually transduced with the HSV-tk gene. This so-called xe2x80x9cbystanderxe2x80x9d cytocidal effect provides an important therapeutic advantage, as it avoids the need to transduce 100% of the tumor cells with the HSV-tk gene. For a detailed description of the bystander effect, see, e.g., Vrionis et al. (1995) J. Neurosurg. 83:698-704, Ishii et al. (1994) J. Cell Biochem. 18A:226, and Freeman et al. (1993) Cancer Res. 53:5274-5283.
In vivo transfer of drug-susceptibility genes is especially suited for treating solid tumors that are growing rapidly and invading normal tissue composed largely of nonproliferating or quiescent cells. Such therapies have thus been applied to the treatment of hepatocellular carcinoma (HCC). HCC is a common human malignancy that is particularly refractive to conventional cancer therapies. Modifications in conventional chemotherapeutic protocols, such as intrahepatic artery infusion of cytotoxic drugs, are able to improve tumor responses but fail to substantially improve patient prognosis or survival. Venook, A. P. (1994) J. Clin. Oncol. 12:1323-1334, Farmer et al. (1994) Cancer 73:2669-2670. The most effective approach to date in the treatment of HCC entails complete surgical ablation of the tumor by partial hepatectomy or by total hepatectomy coupled with liver transplantation.
Recently, investigators have shown suppression of tumor growth and increased survival rates in transgenic murine subjects that express HSV-tk in HCC cells when those subjects were treated with ganciclovir. Macri et al. (1994) Hum. Gene Ther. 5:175-182. Retroviral vehicles have been used to transfer varicella-zoster virus thymidine kinase into HCC tumor cells to confer sensitivity to 6-methoxypurine arabino-nucleoside. Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043. Further, adenoviral vehicles have been used to transfer HSV-tk into HCC cells to confer sensitivity to ganciclovir. Qian et al. (1995) Hepatology 22:118-123.
The use of replication-deficient retroviral vectors to transduce the HSV-tk gene into solid tumor cells is also being clinically investigated as a new approach in the treatment of human ovarian cancer. Ishii et al. (1994) J. Cell Biochem. 18A:226. Additionally, studies have been described wherein pancreatic cancer xenografts were successfully treated in severe combined immunodeficient (scid) mice using retrovirally-mediated HSV-tk transduction and ganciclovir treatment. DiMaio et al. (1994) Surgery 116:205-213. Retroviral vectors have also been used to transduce lymphoma, fibrosarcoma and adenocarcinoma cells with the HSV-tk gene in culture and in vivo, rendering those cells conditionally sensitive to ganciclovir. Plautz et al. (1991) New Biol. 3:709-715, Freeman et al. (1991) Federal Register 56 #138, p. 33174, Moolten et al. (1990) Hum. Gene Ther. 1:125-134, Moolten, F. L. (1986) Cancer Res. 46:5276-5281.
Drug sensitivity therapies are also being investigated in the treatment of malignant melanoma. The incidence of malignant melanoma in the United States continues to increase at a rate of about 2-3% annually, resulting in increased morbidity and mortality as a result of this disease. This serious health problem is even further exacerbated, as an effective treatment for melanoma has remained elusive due to a high propensity for metastatic spread and the resistance of such tumor cells to the most widely used chemotherapeutic regimes. Accordingly, new alternative therapies in the treatment of malignant melanoma include sensitizing melanoma cells to ganciclovir by transducing those cells with HSV-tk via an adenoviral-based gene delivery system. Bonnekoh et al. (1995) J. Investigative Dermatology 104:313-317.
A great deal of interest has also developed around providing alternative therapeutic techniques for the treatment of malignant brain tumors. Brain tumors, e.g., malignant primary intracranial tumors or metastatic tumors, are rapidly debilitating and extremely lethal forms of cancer. The most common primary intracranial tumors are malignant gliomas which account for about 30-40% of primary brain tumors in adults. Patients presenting with glioblastoma multiforme, a highly malignant form of glioma, have an average life expectancy of less than about one year despite a number of recent improvements in neurosurgical techniques and neuroradiological imaging modalities. In light of this poor prognosis, and the inability of current therapeutic approaches (e.g., surgical resection, irradiation and chemotherapy) to effectively treat malignant gliomas, drug sensitization therapy, such as the in vivo transduction of glioma cells with HSV-tk, may provide a new therapeutic approach in the treatment of intracranial solid tumors.
In particular, several methods have been developed for transducing glioma cells with HSV-tk. One method involves in situ inoculation of a brain tumor mass with packaging cells capable of producing replication-defective retroviral particles carrying the HSV-tk gene, followed by treatment with ganciclovir. Ram et al. (1994) J. Neurosurg. 81:256-260, Culver et al. (1992) Science 256:1550-1552, Kim et al. (1991) J. Neurosurg. 74:27-37, Short et al. (1990) J. Neurosci. Res. 27:427-439. The retroviral particles are secreted from the inoculated packaging cells to transduce local tumor cells, rendering them sensitive to the ganciclovir pro-drug. Another approach involves intra-tumoral injection with recombinant adenovirus vectors to transduce malignant glioma cells with HSV-tk, coupled with ganciclovir treatment. Badie et al. (1994) Neurosurgery 35:910-916, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057, Perez-Cruet et al. (1994) J. Neurosci Res. 39:506-511. Yet another method involves intra-tumoral injection of genetically engineered herpes simplex virus (HSV) particles into solid brain tumors. Chambers et al. (1995) Proc. Natl. Acad. Sci. USA 92:1411-1415, Markert et al. (1993) Neurosurgery 32:597-603, Takamiya et al. (1993) J. Neurosurg. 79:104-10, Martuza et al. (1991) Science 252:854-856.
Although drug sensitization techniques, such as the above-described HSV-tk transduction therapies, have shown promise in the treatment of cancer, such approaches have not yet fulfilled their theoretical potential. This shortcoming may be due in part to the low transduction efficiency of retroviral vectors. Furthermore, the pathogenicity of retroviral and adenoviral particles limits the use of such particles in developing safe and effective gene delivery systems for use in human subjects.
The shortcomings of prior retroviral and adenoviral-based suicide gene delivery systems could be overcome in part by developing vectors that provide an ancillary therapeutic effect in conjunction with drug sensitization. In this regard, gene transfer systems could be used to also increase the immunogenicity of transduced tumor cells, leading to a local and/or systemic antitumor effect that is not dependent on the administration of a chemotherapeutic agent. The immunogenicity of the transduced cells could be increased using gene transfer to cause local cytokine production or enhance the expression of major histocompatibility complex antigen expression. Gansbacher et al. (1990) Cancer Res. 50:7820, Tepper et al. (1989) Cell 57:503, Itaya et al. (1987) Cancer Res. 47:3136. Alternatively, gene transfer systems could be used to transduce tumor cells with tumor suppressor genes, alone or in conjunction with a drug-sensitizing gene or a cytokine gene. Despite the advantages of such systems, gene delivery systems that combine multiple antitumor strategies in a single gene delivery vector have not heretofore been described.
Further, limitations due to pathogenic or immunogenic characteristics of the adenoviral or retroviral vectors could be avoided by using an alternative gene delivery system. In this regard, even though retroviral vectors are able to mediate stable, integrated gene transfer in actively dividing cells which provides for enhanced selectivity in the context of treating some neoplastic disease (Miller et al. (1990) Mol. Cell Biol. 10:4239-4242), these systems suffer from several serious drawbacks. For example, the inability to transduce non-dividing or slowly-dividing cells lowers the ability of such systems to treat solid tumors where only a portion of the cells are proliferating at one time. Replication-incompetent retroviral gene delivery systems are also known to be inefficient at gene transfer, often failing to transduce cells at distances of more than a few millimeters from an injection site. The use of retroviral particles in gene delivery is also hampered by the inability to produce substantial viral titers.
The use of adenoviral vectors in gene delivery avoids a number of the problems associated with retroviral-based systems. Adenoviruses can be produced in high titers, and are able to infect quiescent as well as replicating target cells. Despite these advantages, adenovirus vector systems still have several drawbacks which limit their effectiveness in gene delivery. Most significantly, high dose intracerebral injection of adenoviral vectors alone has been shown to produce a direct cytotoxic (neurotoxic) effect, and synergistic toxicity has been observed when such injections are coupled with ganciclovir (GCV) administration. Goodman et al. (1996) Hum Gene Therapy 7:1241-1250. These results compel caution in the clinical use of recombinant adenovirus. Adenovirus vectors also express viral proteins that may elicit a strong non-specific immune response in the host. This non-specific immune reaction may increase toxicity or preclude subsequent treatments because of humoral and/or T cell responses against the adenoviral particles.
Thus, there remains a need to provide an alternative approach of sensitizing solid tumor cells using a gene delivery method that avoids the problems associated with prior retroviral and adenoviral vector-based systems. The method should also be capable of providing an ancillary therapeutic effect to increase the efficacy of the therapeutic method. An ancillary therapeutic effect could be provided by increasing the immune recognition of a transduced tumor cell by the host immune cells. Alternatively, an ancillary effect could be effected using tumor suppressor genes to provide a cytostatic effect in the transduced tumor cells. One particularly attractive alternative would entail the use of adeno-associated virus (AAV) gene delivery systems.
Recombinant vectors based on AAV particles have been used for DNA delivery. AAV is a helper-dependent DNA parvovirus which belongs to the genus Dependovirus. AAV has a wide host range and is able to replicate in cells from any species so long as there is also a successful infection of such cells with a suitable helper virus. AAV has not been associated with any human or animal disease. For a review of AAV, see, e.g., Berns and Bohenzky (1987) Advances in Virus Research (Academic Press, Inc.) 32:243-307.
The construction of recombinant vectors based on AAV has been described. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Numbers WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.
Recombinant AAV vectors are capable of transducing several cell types, including hematopoietic cells, respiratory epithelial cells (Flotte et al. (1992) Am. J. Respir. Cell Mol. Biol. 7:349-356; Flotte et al. (1993) J. Biol. Chem. 268:3781-3790; Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617) and neurons of the central nervous system (Kaplitt et al. (1994) Nature Genetics 8:148-154). These cell types are well-differentiated, slowly-dividing or postmitotic. Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617; Kaplitt et al. (1994) Nature Genetics 8:148-154.
A recombinant AAV-based gene transfer system has been described for the transduction of HSV-tk into cells of the central or peripheral nervous systems in a mammalian subject to render those cells sensitive to ganciclovir. See, International Publication No. WO 95/28493. This system is particularly designed for use in the treatment of neurological disorders such as Parkinson""s disease and in the treatment of brain tumors. Even though this system may avoid problems associated with prior adenoviral or retroviral-based systems, it fails to provide an ancillary therapeutic effect, thereby reducing its overall effectiveness.
Accordingly, there remains a need to provide an AAV-based gene delivery system that is capable of transducing a wide range of solid cancer cells to render them sensitive to selected compounds or compositions, wherein the system also provides an ancillary therapeutic effect. Particularly, there remains a need to provide a multi-faceted delivery system that increases the immune recognition of a transduced tumor cell by the host immune cells in conjunction with providing drug susceptibility. There also remains a need to provide a system which provides a cytostatic effect in a transduced, drug-susceptible tumor cell.
It is a primary object of the present invention to provide an AAV-based gene delivery system for transducing solid cancer cells with a drug sensitizing gene, wherein the system also provides for an ancillary therapeutic effect. The system is capable of being used to transduce a wide range of different tumor cell types, and is thus useful in the treatment of a variety of neoplastic diseases. The ancillary therapeutic effect serves to increase the overall efficacy of the present therapeutic system, and is provided by either increasing a local immune response to transduced tumor cells by the host immune cells or, imparting a cytostatic and/or cytotoxic effect to the transduced cell. The underlying AAV particles avoid a number of the problems encountered with prior retroviral- or adenoviral-based gene delivery systems.
Accordingly, in one embodiment, the invention relates to a method of simultaneously delivering a first gene and a second gene to a target solid tumor cell (i.e., transducing the target cell). Particularly, a recombinant adeno-associated virus (rAAV) virion is provided which includes a first gene that is capable of being expressed to provide a transduced target cell with enhanced susceptibility to a selected cytotoxic agent. The rAAV virion also includes a second gene that is capable of providing an ancillary therapeutic effect to the transduced cell. The first gene and the second gene are operably linked to control elements that are capable of directing the in vivo transcription and translation of those genes in the transduced cell. Each gene may be associated with a discrete set of control elements, or both genes can be associated with a single group of control elements.
In one aspect of the invention, the second gene is capable of being expressed by the transduced tumor cell to enhance the immunogenicity of the transduced cell. Thus, the second gene can encode a cytokine, such as type I interferons, tumor necrosis factor (TNF), interleukin-2 (IL-2), gamma interferon (IFN-xcex3), lymphotoxin, interleukin-12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Alternatively, the second gene is capable of being expressed to provide a tumorstatic effect in the transduced cell. In this regard, the second gene can be a tumor suppressor gene, such as p53, RB1, WT1, NF1, VHL, and APC.
In another embodiment of the invention, a method is provided for treating neoplastic disease in a mammalian subject. The method entails the steps of: (1) transducing a solid tumor cell of a mammalian subject in vivo using a therapeutically effective amount of a pharmaceutical composition which comprises (a) a pharmaceutically acceptable excipient, and (b) rAAV virions, where the rAAV virions comprise an AAV vector that includes a first gene that is capable of being expressed to provide the transduced tumor cell with enhanced susceptibility to a selected cytotoxic agent, a second gene that is capable of providing an ancillary therapeutic effect, and control elements operably linked to the first gene and the second gene such that the control elements are capable of directing the in vivo transcription and translation of the first gene and the second gene when they are present in the mammalian subject; and then (2) administering a therapeutically effective amount of the selected cytotoxic agent to the mammalian subject.
As described above, the second gene of the rAAV virion can be used to enhance the immunogenicity of the transduced cell or to provide a tumorstatic effect in the transduced cell. Thus, the second gene can encode a cytokine (e.g., alpha interferon (IFN-xcex1), beta interferon (IFN-xcex2), gamma interferon (IFN-xcex3), tumor necrosis factor (TNF), interleukin-2 (IL-2), lymphotoxin, interleukin-12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), or the second gene can be a tumor suppressor gene (e.g., p53, RB1, WT1, NF1, VHL, and APC).
In other embodiments, the invention is directed to the provision of an AAV vector which includes a drug susceptibility gene and a second gene, wherein the second gene either encodes a cytokine or is a tumor suppressor gene. The first gene and the second gene are operably linked to control elements capable of directing the in vivo transcription and translation thereof. The invention is also directed to a recombinant AAV virion containing the subject vector.
In yet another embodiment of the invention, AAV vectors are provided which include a single gene, such as an interferon gene or a tumor suppressor gene. The AAV vectors can be used to provide either an immunogenic or a tumorstatic effect in a transduced solid tumor cell.
These and other embodiments of the invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.